{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,12,24]],"date-time":"2025-12-24T11:39:44Z","timestamp":1766576384803,"version":"3.48.0"},"reference-count":46,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2025,12,24]],"date-time":"2025-12-24T00:00:00Z","timestamp":1766534400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Ordos Science & Technology Plan","award":["YF20240007"],"award-info":[{"award-number":["YF20240007"]}]},{"name":"Special Project for the Construction of Scientific Research and Innovation Teams, Class B Team","award":["BR251303"],"award-info":[{"award-number":["BR251303"]}]},{"name":"Inner Mongolia Autonomous Region First Class Discipline Research Special Project","award":["YLXKZX-NND-012"],"award-info":[{"award-number":["YLXKZX-NND-012"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pathogens"],"abstract":"<jats:p>Paratuberculosis (PTB), caused by Mycobacterium avium subsp. paratuberculosis (MAP), is a chronic intestinal disease in ruminants. PTB is difficult to diagnose, control, and eradicate, leading to substantial economic losses. Thus, sensitive and specific detection methods are urgently required. crRNA and primers targeting the MAP ATPase FtsK gene were designed for recombinase polymerase amplification (RPA) and nested PCR. Fecal DNA was amplified using RPA or nested PCR, purified with Tris-saturated phenol-chloroform-isoamyl alcohol, and detected via CRISPR-Cas12a. Moreover, signals were read using a qPCR instrument, fluorescence reader, or lateral flow strips. RPA\u2013CRISPR-Cas12a and nested PCR\u2013CRISPR-Cas12a assays were optimized and validated on 50 clinical samples and 7 MAP cultures. The limits of detection were 1 \u00d7 10\u221210 \u03bcg\/\u03bcL for RPA\u2013CRISPR-Cas12a and 1 \u00d7 10\u221214 \u03bcg\/\u03bcL for nested PCR\u2013CRISPR-Cas12a. Efficient cleavage of the ssDNA reporter occurred at DNA concentrations of \u22651 \u00d7 10\u22124 \u03bcg\/\u03bcL, producing a strong fluorescent signal. All three detection methods showed perfect agreement with reference assays across both sample sets. This study presents the first integration of RPA or nested PCR with CRISPR-Cas12a for MAP detection, enabling rapid, specific, and highly sensitive diagnosis. Flexible detection options allow adaptation to available resources and bacterial loads, supporting practical use in PTB control.<\/jats:p>","DOI":"10.3390\/pathogens15010024","type":"journal-article","created":{"date-parts":[[2025,12,24]],"date-time":"2025-12-24T11:22:14Z","timestamp":1766575334000},"page":"24","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Rapid and Simple Detection of Mycobacterium avium subsp. paratuberculosis Using a Lateral Flow Assay Based on CRISPR-Cas12a Combined with Recombinase Polymerase Amplification or Nested PCR"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0009-0001-1337-3901","authenticated-orcid":false,"given":"Yue-Rong","family":"Lv","sequence":"first","affiliation":[{"name":"College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010018, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yi-Yang","family":"Liu","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010018, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rong","family":"Zhang","sequence":"additional","affiliation":[{"name":"Otok Banner Animal Disease Prevention and Control Center, Ordos 016100, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Bo","family":"Yang","sequence":"additional","affiliation":[{"name":"Animal Disease Control Center of Ordos, Ordos 016100, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0000-1640-7890","authenticated-orcid":false,"given":"Shi-Yuan","family":"Xue","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010018, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yu-Lin","family":"Ding","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010018, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jun-Tao","family":"Jia","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010018, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hasi","family":"Bayaer","sequence":"additional","affiliation":[{"name":"Otok Banner Animal Disease Prevention and Control Center, Ordos 016100, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Alateng","family":"Bagen","sequence":"additional","affiliation":[{"name":"Otok Banner Animal Disease Prevention and Control Center, Ordos 016100, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rui-Bin","family":"Chen","sequence":"additional","affiliation":[{"name":"Otok Banner Animal Disease Prevention and Control Center, Ordos 016100, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Siqin","family":"Tunala","sequence":"additional","affiliation":[{"name":"Otok Banner Animal Disease Prevention and Control Center, Ordos 016100, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8538-5605","authenticated-orcid":false,"given":"Li","family":"Zhao","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010018, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4432-9433","authenticated-orcid":false,"given":"Yong-Hong","family":"Liu","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010018, China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,12,24]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Xue, S.Y., Ma, W., Li, M.Y., Meng, W.K., Ding, Y.L., Yang, B., Lv, Y.R., Chen, R.B., Wu, Z.H., and Tunala, S. 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